Non-invasive diagnosis of graft rejection in organ transplant patients

ABSTRACT

The invention provides methods, devices, compositions and kits for diagnosing or predicting transplant status or outcome in a subject who has received a transplant.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 17/323,917, filed May 18, 2021, which is a Continuation ofapplication Ser. No. 16/361,752, filed Mar. 22, 2019, now U.S. Pat. No.11,098,350, issued Aug. 24, 2021, which is a Continuation of applicationSer. No. 15/788,549 filed Oct. 19, 2017, now abandoned, which is aContinuation of application Ser. No. 14/188,455 filed Feb. 24, 2014, nowU.S. Pat. No. 9,845,497, issued Dec. 19, 2019, which is a Continuationof application Ser. No. 13/508,318 filed Jul. 19, 2012, now patented asU.S. Pat. No. 8,703,652 issued Apr. 22, 2014, which is a 371 applicationand claims the benefit of PCT Application No. PCT/US2010/055604, filedNov. 5, 2010, which claims benefit of U.S. Provisional PatentApplication No. 61/280,674, filed Nov. 6, 2009, which applications areincorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contracts HL099995and OD000251 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith in a text file,S09-367_STAN-706CON13_SEQLIST created on Jun. 9, 2022, and having a sizeof 1,058 bytes. The contents of the text file are incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Organ transplantation is an important medical procedure which saveslives in cases where a patient has organ failure or disablement, and itis now possible to transplant many organs including heart, lungs,kidney, and liver. In some cases, the transplanted organ is rejected bythe recipient patient, which creates a life-threatening situation.Monitoring the patient for rejection is difficult and expensive, oftenrequiring invasive procedures. Furthermore, current surveillance methodslack adequate sensitivity.

The present invention resolves these problems by providing non-invasivemethods of monitoring organ transplant patients for rejection that aresensitive, rapid and inexpensive.

SUMMARY OF THE INVENTION

The invention provides methods, devices, compositions and kits fordiagnosing and/or predicting transplant status or outcome in a subjectwho has received a transplant. In some embodiments, the inventionprovides methods of diagnosing or predicting transplant status oroutcome comprising the steps of: (i) providing a sample from a subjectwho has received a transplant from a donor; (ii) determining thepresence or absence of one or more nucleic acids from the donortransplant, where the one or more nucleic acids from the donor areidentified based on a predetermined marker profile; and (iii) diagnosingor predicting transplant status or outcome based on the presence orabsence of the one or more nucleic acids.

In some embodiments, the transplant status or outcome comprisesrejection, tolerance, non-rejection based allograft injury, transplantfunction, transplant survival, chronic transplant s injury, or titerpharmacological immunosuppression. In some embodiments, thenon-rejection based allograft injury is selected from the group ofischemic injury, virus infection, peri-operative ischemia, reperfusioninjury, hypertension, physiological stress, injuries due to reactiveoxygen species and injuries caused by pharmaceutical agents.

In some embodiments, the sample is selected from the group consisting ofblood, serum, urine, and stool. In some embodiments, the marker profileis a polymorphic marker profile. In some embodiments, the polymorphicmarker profile comprises one or more single nucleotide polymorphisms(SNP's), one or more restriction fragment length polymorphisms (RFLP's),one or more short tandem repeats (STRs), one or more variable number oftandem repeats (VNTR's), one or more hypervariable regions, one or moreminisatellites, one or more dinucleotide repeats, one or moretrinucleotide repeats, one or more tetranucleotide repeats, one or moresimple sequence repeats, or one or more insertion elements. In someembodiments, the polymorphic marker profile comprises one or more SNPs

In some embodiments, the marker profile is determined by genotyping thetransplant donor. In some embodiments, the methods further comprisegenotyping the subject receiving the transplant. In some embodiments,the methods further comprise establishing a profile of markers, wherethe markers are distinguishable between the transplant donor and thesubject receiving the transplant. In some embodiments, the genotyping isperformed by a method selected from the group consisting of sequencing,nucleic acid array and PCR.

In any of the embodiments described herein, the transplant graft maybeany solid organ and skin transplant. In some embodiments, the transplantis selected from the group consisting of kidney transplant, hearttransplant, liver transplant, pancreas transplant, lung transplant,intestine transplant and skin transplant.

In some embodiments, the nucleic acid is selected from the groupconsisting of double-stranded DNA, single-stranded DNA, single-strandedDNA hairpins, DNA/RNA hybrids, RNA and RNA hairpins. In someembodiments, the nucleic acid is selected from the group consisting ofdouble-stranded DNA, single-stranded DNA and cDNA. In some embodiments,the nucleic acid is mRNA. In some embodiments, the nucleic acid isobtained from circulating donor cells. In some embodiments, the nucleicacid is circulating cell-free DNA.

In some embodiments, the presence or absence of the one or more nucleicacids is determined by a method selected from the group consisting ofsequencing, nucleic acid array and PCR. In some embodiments, thesequencing is shotgun sequencing. In some embodiments, the array is aDNA array. In some embodiments, the DNA array is a polymorphism array.In some embodiments, the polymorphism array is a SNP array.

In some embodiments, the methods further comprise quantitating the oneor more nucleic acids. In some embodiments, the amount of the one ormore nucleic acids is indicative of transplant status or outcome. Insome embodiments, the amount of the one or more nucleic acids above apredetermined threshold value is indicative of a transplant status oroutcome. In some embodiments, the threshold is a normative value forclinically stable post-transplantation patients with no evidence oftransplant rejection or other pathologies. In some embodiments, thereare different predetermined threshold values for different transplantoutcomes or status. In some embodiments, temporal differences in theamount of the one or more nucleic acids are indicative of a transplantstatus or outcome.

In some embodiments, the methods described herein have at least 56%sensitivity. In some embodiments, the methods described herein have atleast 78% sensitivity. In some embodiments, the methods described hereinhave a specificity of about 70% to about 100%. In some embodiments, themethods described herein have a specificity of about 80% to about 100%.In some embodiments, the methods described herein a specificity of about90% to about 100%. In some embodiments, the methods described hereinhave a specificity of about 100%.

In some embodiments, the invention provides computer readable mediumscomprising: a set of instructions recorded thereon to cause a computerto perform the steps of: (i) receiving data from one or more nucleicacids detected in a sample from a subject who has received transplantfrom a donor, where the one or more nucleic acids are nucleic acids fromthe donor transplant, and where the one or more nucleic acids from thedonor are identified based on a predetermined marker profile; and (ii)diagnosing or predicting transplant status or outcome based on thepresence or absence of the one or more nucleic acids.

In some embodiments, the invention provides reagents and kits thereoffor practicing one or more of the methods described herein.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 shows patient survival after diagnosis of CAV.

FIG. 2 shows detection of donor DNA in patients receiving gendermismatched transplants.

FIG. 3 shows a time course study for detection of donor DNA in atransplant patient that received a gender mismatched transplant andsuffered a 3A rejection episode.

FIG. 4 shows a time course study for detection of donor DNA in atransplant patient that received a gender mismatched transplant andsuffered a 3A rejection episode.

FIG. 5 depicts in one embodiments of the invention a general strategy tomonitor all transplant patients

FIG. 6A-6B shows sequencing results comparing four levels ofsubstitutions of donor DNA into recipient DNA.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to particularly preferredembodiments of the invention. Examples of the preferred embodiments areillustrated in the following Examples section.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entirety.

Methods, devices, compositions and kits are provided for diagnosing orpredicting transplant status or outcome in a subject who has received atransplant. The transplant status or outcome may comprise rejection,tolerance, non-rejection based transplant injury, transplant function,transplant survival, chronic transplant injury, or titer pharmacologicalimmunosuppression.

This invention describes sensitive and non-invasive methods, devices,compositions and kits for monitoring organ transplant patients, and/orfor diagnosing or predicting transplant status or outcome (e.g.transplant rejection). In some embodiments, the methods, devices,compositions and kits are used to establish a genotype for both thedonor and the recipient before transplantation to enable the detectionof donor-specific nucleic acids such as DNA or RNA in bodily fluids suchas blood or urine from the organ recipient after transplantation.

In some embodiments, the invention provides methods of determiningwhether a patient or subject is displaying transplant tolerance. Theterm “transplant tolerance” includes when the subject does not reject agraft organ, tissue or cell(s) that has been introduced into/onto thesubject. In other words, the subject tolerates or maintains the organ,tissue or cell(s) that has been transplanted to it. The term “patient”or “subject” as used herein includes humans as well as other mammals.

In some embodiments the invention provides methods for diagnosis orprediction of transplant rejection. The term “transplant rejection”encompasses both acute and chronic transplant rejection. “Acuterejection or AR” is the rejection by the immune system of a tissuetransplant recipient when the transplanted tissue is immunologicallyforeign. Acute rejection is characterized by infiltration of thetransplanted tissue by immune cells of the recipient, which carry outtheir effector function and destroy the transplanted tissue. The onsetof acute rejection is rapid and generally occurs in humans within a fewweeks after transplant surgery. Generally, acute rejection can beinhibited or suppressed with immunosuppressive drugs such as rapamycin,cyclosporin A, anti-CD40L monoclonal antibody and the like.

“Chronic transplant rejection or CR” generally occurs in humans withinseveral months to years after engraftment, even in the presence ofsuccessful immunosuppression of acute rejection. Fibrosis is a commonfactor in chronic rejection of all types of organ transplants. Chronicrejection can typically be described by a range of specific disordersthat are characteristic of the particular organ. For example, in lungtransplants, such disorders include fibroproliferative destruction ofthe airway (bronchiolitis obliterans); in heart transplants ortransplants of cardiac tissue, such as valve replacements, suchdisorders include fibrotic atherosclerosis; in kidney transplants, suchdisorders include, obstructive nephropathy, nephrosclerorsis,tubulointerstitial nephropathy; and in liver transplants, such disordersinclude disappearing bile duct syndrome. Chronic rejection can also becharacterized by ischemic insult, denervation of the transplantedtissue, hyperlipidemia and hypertension associated withimmunosuppressive drugs.

In some embodiments, the invention further includes methods fordetermining an immunosuppressive regimen for a subject who has receiveda transplant, e.g., an allograft.

Certain embodiments of the invention provide methods of predictingtransplant survival in a subject that has received a transplant. Theinvention provides methods of diagnosing or predicting whether atransplant in a transplant patient or subject will survive or be lost.In certain embodiments, the invention provides methods of diagnosing orpredicting the presence of long-term graft survival. By “long-term”graft survival is meant graft survival for at least about 5 years beyondcurrent sampling, despite the occurrence of one or more prior episodesof acute rejection. In certain embodiments, transplant survival isdetermined for patients in which at least one episode of acute rejectionhas occurred. As such, these embodiments provide methods of determiningor predicting transplant survival following acute rejection. Transplantsurvival is determined or predicted in certain embodiments in thecontext of transplant therapy, e.g., immunosuppressive therapy, whereimmunosuppressive therapies are known in the art. In yet otherembodiments, methods of determining the class and/or severity of acuterejection (and not just the presence thereof) are provided.

In some embodiments, the invention provides methods for diagnosis orprediction of non-rejection based transplant injury. Examples ofnon-rejection based graft injury include, but are not limited to,ischemic injury, virus infection, peri-operative ischemia, reperfusioninjury, hypertension, physiological stress, injuries due to reactiveoxygen species and injuries caused by pharmaceutical agents.

As in known in the transplantation field, the transplant organ, tissueor cell(s) may be allogeneic or xenogeneic, such that the grafts may beallografts or xenografts. A feature of the graft tolerant phenotypedetected or identified by the subject methods is that it is a phenotypewhich occurs without immunosuppressive therapy, i.e., it is present in ahost that is not undergoing immunosuppressive therapy such thatimmunosuppressive agents are not being administered to the host. Thetransplant graft maybe any solid organ and skin transplant. Examples oforgan transplants that can be analyzed by the methods described hereininclude but are not limited to kidney transplant, pancreas transplant,liver transplant, heart transplant, lung transplant, intestinetransplant, pancreas after kidney transplant, and simultaneouspancreas-kidney transplant.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Introduction

Methods, devices, compositions and kits are provided for diagnosing orpredicting transplant status or outcome in a subject who has received atransplant.

As mention above, monitoring transplant patients for transplant statusor outcome is difficult and expensive, often requiring non-sensitive andinvasive procedures. For instance, in heart transplant patients acuterejection surveillance requires serial endomyocardial biopsies that areroutinely performed at weekly and monthly intervals during the initialyear after transplant, with a total of 6-8 biopsies in most patients.Advances in immunosuppression, rejection surveillance, and earlyrecognition and treatment of life-threatening infections have led tocontinuous improvements in early outcomes after cardiac transplantation.(Taylor, D. O., et al., J Heart Lung Transplant, 27, 943-956 (2008))However, there has not been a similar improvement in late mortality,which is largely attributable to cardiac allograft vasculopathy (CAV).(FIG. 1) Today, CAV remains the major cause of late graft failure anddeath amongst the nearly 22,000 living heart transplant recipients inthe United States. Early detection of CAV, prior to the development ofangiographically apparent disease, graft dysfunction, or symptom onsetis important because patient mortality after detection by coronaryangiography (the standard of care) is unacceptably high, with 2-yearmortality rates of 50% having been reported. Current surveillancemethods for CAV lack adequate sensitivity or require invasive proceduresand the most commonly applied method, coronary angiography, lackssensitivity (Kobashigawa, J. A., et al., J Am Coll Cardiol, 45,1532-1537 (2005)). Delayed diagnosis due to underestimation of diseaseseverity is a feature of coronary angiography that is largely overcomeby intravascular ultrasound (IVUS). (Fitzgerald, P. J., et al.,Circulation, 86, 154-158 (1992)) However, both of these invasiveleft-heart, arterial catheter methods are costly, resource intensive,and associated with significant risk of morbidity and patientdiscomfort. Early detection of CAV, prior to the development ofangiographically apparent disease, graft dysfunction, or symptom onsetis crucial to guide the appropriate use of emerging therapies thatretard and occasionally reverse progression of CAV. The development ofmarkers for early, non-invasive, safe, and cost-effective detection ofacute rejection and CAV, and their rapid translation to a practical andreliable test that can be used in the clinic represents a major unmetmedical need for the nearly 22,000 living heart transplant recipients inthe United States, and a similar number worldwide.

The pressing need for early diagnosis and risk stratification is furtherunderscored by recent studies demonstrating delayed progression and/orreversal of CAV following intervention with newer immunosuppressiveregimens. Since the use of these newer therapies are encumbered byadverse effects, drug interactions, and cost, it is important toidentity the patients in whom the benefits outweigh the risks. Asidefrom its impact on mortality and morbidity, CAV surveillance is costlyin terms of resource utilization and potential for patientcomplications. Given the current standard of care to perform annualcoronary angiography for the initial five years after hearttransplantation, each patient surviving to year 5 will have received 4angiograms for an average fully loaded cost of $25,000 per angiogram.Since the 5-year survival rate after heart transplantation is 72%,approximately 1,440 patients out of the 2,000 patients receiving hearttransplants each year will undergo 4 procedures for a total of at least5,760 procedures. At an average cost of $25,000 per coronary angiogram,this will amount to $144,000,000 per year in healthcare dollars formonitoring patients after heart transplantation. A non-invasive testthat identifies the patients at low risk of CAV would mean that coronaryangiography could be safely avoided in this group, thereby considerablyreducing the cost of their long-term management.

The same difficulties and expenses are experienced by patients receivingother type of transplants.

a. Circulating Nucleic Acids

Circulating, or cell-free, DNA was first detected in human blood plasmain 1948. (Mandel, P. Metais, P., C R Acad. Sci. Paris, 142, 241-243(1948)) Since then, its connection to disease has been established inseveral areas. (Tong, Y. K. Lo, Y. M., Clin Chim Acta, 363, 187-196(2006)) Studies reveal that much of the circulating nucleic acids inblood arise from necrotic or apoptotic cells (Giacona, M. B., et al.,Pancreas, 17, 89-97 (1998)) and greatly elevated levels of nucleic acidsfrom apoptosis is observed in diseases such as cancer. (Giacona, M. B.,et al., Pancreas, 17, 89-97 (1998); Fournie, G. J., et al., Cancer Lett,91, 221-227 (1995)) Particularly for cancer, where the circulating DNAbears hallmark signs of the disease including mutations in oncogenes,microsatellite alterations, and, for certain cancers, viral genomicsequences, DNA or RNA in plasma has become increasingly studied as apotential biomarker for disease. For example, Diehl et al recentlydemonstrated that a quantitative assay for low levels of circulatingtumor DNA in total circulating DNA could serve as a better marker fordetecting the relapse of colorectal cancer compared withcarcinoembryonic antigen, the standard biomarker used clinically.(Diehl, F., et al., Proc Natl Acad Sci, 102, 16368-16373 (2005); Diehl,F., et al., Nat Med, 14, 985-990 (2008)) Maheswaran et al reported theuse of genotyping of circulating cells in plasma to detect activatingmutations in epidermal growth factor receptors in lung cancer patientsthat would affect drug treatment. (Maheswaran, S., et al., N Engl J Med,359, 366-377 (2008)) These results collectively establish bothcirculating DNA, either free in plasma or from circulating cells, as auseful species in cancer detection and treatment. Circulating DNA hasalso been useful in healthy patients for fetal diagnostics, with fetalDNA circulating in maternal blood serving as a marker for gender, rhesusD status, fetal aneuploidy, and sex-linked disorders. Fan et al recentlydemonstrated a strategy for detecting fetal aneuploidy by shotgunsequencing of cell-free DNA taken from a maternal blood sample, amethodology that can replace more invasive and risky techniques such asamniocentesis or chorionic villus sampling. (Fan, H. C., Blumenfeld, Y.J., Chitkara, U., Hudgins, L., Quake, S. R., Proc Natl Acad Sci, 105,16266-16271 (2008))

In all these applications of circulating nucleic acids, the presence ofsequences differing from a patient's normal genotype has been used todetect disease. In cancer, mutations of genes are a tell-tale sign ofthe advance of the disease; in fetal diagnostics, the detection ofsequences specific to the fetus compared to maternal DNA allows foranalysis of the health of the fetus.

In some embodiments, the invention provides non-invasive diagnosticsexists for organ transplant patients where sequences from the organdonor, otherwise “foreign” to the patient, can be quantitatedspecifically. Without intending to be limited to any theory, ascell-free DNA or RNA often arises from apoptotic cells, the relativeamount of donor-specific sequences in circulating nucleic acids shouldprovide a predictive measure of on-coming organ failure in transplantpatients for many types of solid organ transplantation including, butnot limited to, heart, lung, liver, and kidney.

b. Circulating Nucleic Acids and Transplant Rejection

In some embodiments, the invention provides methods, devices,compositions and kits for detection and/or quantitating circulatingnucleic acids, either free in plasma or from circulating cells, for thediagnosis, prognosis, detection and/or treatment of a transplant statusor outcome. There have been claims of detection of donor-DNA insex-mismatched liver and kidney transplant patients; conventional PCRwas used to search for Y chromosome sequences from male donors in theblood of female patients. (Lo, Y. M., et al., Lancet, 351, 1329-1330(1998) However, in a follow-on study Y-chromosome specific sequenceswere not detected above background in 16 out of 18 patients using a moreaccurate quantitative polymerase chain reaction (qPCR) assay. (Lui, Y.Y., et al., Clin Chem, 49, 495-496 (2003)) In renal transplantation,urine samples of similarly sex-mismatched transplant patients wereanalyzed and Y chromosomal DNA was detected in patients immediatelyafter transplantation as well as during graft rejection episodes.(Zhang, J., et al., Clin Chem, 45, 1741-1746 (1999); Zhong, X. Y., etal., Ann N Y Acad Sci, 945, 250-257 (2001))

Example 1 examined gender-mismatched heart transplant recipients andapplied digital PCR (Warren, L., Bryder, D., Weissman, I. L., Quake, S.R., Proc Natl Acad Sci, 103, 17807-17812 (2006); Fan, H. C. Quake, S.R., Anal Chem, 79, 7576-7579 (2007)) to detect the level ofdonor-derived chromosome Y signal in plasma samples taken at the sametime that an endomyocardial biopsy determined a grade 3A or 3B rejectionepisode. While there was not any significant chromosome Y signaldetected from four control female-to-female transplant patients, 1.5-8%total genomic fraction for chromosome Y signals at the rejection timepoints was observed for three male-to-female transplant patients acrossfour rejection episodes (FIG. 2). A time-course study for one of thesepatients revealed that the level of chromosome Y detected in plasma wasneglible in plasma at three months prior to rejection, butincreased >10-fold to 2% of total genomic fraction at the time a biopsydetermined rejection (See FIGS. 3 and 4). Collectively, these resultsestablish that for heart transplant patients, donor-derived DNA presentin plasma can serve as a potential marker for the onset of organfailure.

While each of these studies demonstrates donor-DNA in bodily fluids fordifferent solid organ transplants, they are all limited to the specialcase of females receiving organs from males and will not work forfemales receiving from females, males receiving from males, or malesreceiving from females. Further problems with this strategy arise fromthe prevalence of microchimerism in female patients where past malepregnancies or blood transfusions may lead to Y-chromosome specificsignals from sources other than the transplanted organ. (Hubacek, J. A.,Vymetalova, Y., Bohuslavova, R., Kocik, M., Malek, I., Transplant Proc,39, 1593-1595 (2007); Vymetalova, Y., et al., Transplant Proc, 40,3685-3687 (2008)) The detection of donor-specific human leukocyteantigen (HLA) alleles in circulating DNA has been considered as a signalfor organ rejection, specifically for kidney and pancreas transplantpatients. (Gadi, V. K., Nelson, J. L., Boespflug, N. D., Guthrie, K. A.,Kuhr, C. S., Clin Chem, 52, 379-382 (2006)) However, this strategy willalso be limited by the inability to distinguish HLA alleles between alldonors and recipients, particularly for common HLA types, and thepotential complication of microchimerism such as from bloodtransfusions. (Baxter-Lowe, L. A. Busch, M. P., Clin Chem, 52, 559-561(2006))

In some embodiments, the invention provides a universal approach tononinvasive detection of graft rejection in transplant patients whichcircumvents the potential problems of microchimerism from DNA from otherforeign sources and is general for all organ recipients withoutconsideration of gender. In some embodiments, a genetic fingerprint isgenerated for the donor organ. This approach allows for a reliableidentification of sequences arising solely from the organtransplantation that can be made in a manner that is independent of thegenders of donor and recipient.

In some embodiments, both the donor and recipient will be genotypedprior to transplantation. Examples of methods that can be used togenotyped the transplant donor and the transplant recipient include, butare not limited to, whole genome sequencing, exome sequencing, orpolymorphisms arrays (e.g., SNP arrays). A set of relevant anddistinguishable markers between the two sources is established. In someembodiments, the set of markers comprises a set of polymorphic markers.Polymorphic markers include single nucleotide polymorphisms (SNP's),restriction fragment length polymorphisms (RFLP's), short tandem repeats(STRs), variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. In some embodiments, the set of markers comprises SNPs.

Following transplantation, bodily fluid such as blood can be drawn fromthe patient and analyzed for markers. Examples of bodily fluids include,but are not limited to, smears, sputum, biopsies, secretions,cerebrospinal fluid, bile, blood, lymph fluid, saliva, and urine.Detection, identification and/or quantitation of the donor-specificmarkers (e.g. polymorphic markers such as SNPs) can be performed usingreal-time PCR, chips (e.g., SNP chips), high-throughput shotgunsequencing of circulating nucleic acids (e.g. cell-free DNA), as well asother methods known in the art including the methods described herein.The proportion of donor nucleic acids can be monitored over time and anincrease in this proportion can be used to determine transplant statusor outcome (e.g. transplant rejection).

In some embodiments, where the transplant is a xenotransplant,detection, identification and/or quantitation of the donor-specificmarkers can be performed by mapping one or more nucleic acids (e.g.,DNA) to the genome of the specie use to determine whether the one ormore nucleic acids come from the transplant donor. Polymorphic markersas described above can also be used where the transplant is axenotransplant.

In any of the embodiments described herein, the transplant graft can beany solid organ or skin transplant. Examples of organ transplants thatcan be analyzed by the methods described herein include but are notlimited to kidney transplant, pancreas transplant, liver transplant,heart transplant, lung transplant, intestine transplant, pancreas afterkidney transplant, and simultaneous pancreas-kidney transplant.

Samples

In some embodiments, the methods described herein involve performing oneor more genetic analyses or detection steps on nucleic acids. In someembodiments target nucleic acids are from a sample obtained from asubject that has received a transplant. Such subject can be a human or adomesticated animal such as a cow, chicken, pig, horse, rabbit, dog,cat, or goat. In some embodiments, the cells used in the presentinvention are taken from a patient. Samples derived from an animal,e.g., human, can include, for example whole blood, sweat, tears, saliva,ear flow, sputum, lymph, bone marrow suspension, lymph, urine, saliva,semen, vaginal flow, cerebrospinal fluid, brain fluid, ascites, milk,secretions of the respiratory, intestinal or genitourinary tracts fluid,a lavage of a tissue or organ (e.g. lung) or tissue which has beenremoved from organs, such as breast, lung, intestine, skin, cervix,prostate, pancreas, heart, liver and stomach. For example, a tissuesample can comprise a region of functionally related cells or adjacentcells. Such samples can comprise complex populations of cells, which canbe assayed as a population, or separated into sub-populations. Suchcellular and acellular samples can be separated by centrifugation,elutriation, density gradient separation, apheresis, affinity selection,panning, FACS, centrifugation with Hypaque, etc. By using antibodiesspecific for markers identified with particular cell types, a relativelyhomogeneous population of cells may be obtained. Alternatively, aheterogeneous cell population can be used. Cells can also be separatedby using filters. For example, whole blood can also be applied tofilters that are engineered to contain pore sizes that select for thedesired cell type or class. Cells can be filtered out of diluted, wholeblood following the lysis of red blood cells by using filters with poresizes between 5 to 10 μm, as disclosed in U.S. patent application Ser.No. 09/790,673. Other devices can separate cells from the bloodstream,see Demirci U, Toner M., Direct etch method for microfluidic channel andnanoheight post-fabrication by picoliter droplets, Applied PhysicsLetters 2006; 88 (5), 053117; and Irimia D, Geba D, Toner M., Universalmicrofluidic gradient generator, Analytical Chemistry 2006; 78:3472-3477. Once a sample is obtained, it can be used directly, frozen,or maintained in appropriate culture medium for short periods of time.Methods to isolate one or more cells for use according to the methods ofthis invention are performed according to standard techniques andprotocols well-established in the art.

To obtain a blood sample, any technique known in the art may be used,e.g. a syringe or other vacuum suction device. A blood sample can beoptionally pre-treated or processed prior to enrichment. Examples ofpre-treatment steps include the addition of a reagent such as astabilizer, a preservative, a fixant, a lysing reagent, a diluent, ananti-apoptotic reagent, an anti-coagulation reagent, an anti-thromboticreagent, magnetic property regulating reagent, a buffering reagent, anosmolality regulating reagent, a pH regulating reagent, and/or across-linking reagent.

When a blood sample is obtained, a preservative such an anti-coagulationagent and/or a stabilizer can be added to the sample prior toenrichment. This allows for extended time for analysis/detection. Thus,a sample, such as a blood sample, can be analyzed under any of themethods and systems herein within 1 week, 6 days, 5 days, 4 days, 3days, 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 2 hrs, or 1 hr from the timethe sample is obtained.

In some embodiments, a blood sample can be combined with an agent thatselectively lyses one or more cells or components in a blood sample. Forexample platelets and/or enucleated red blood cells are selectivelylysed to generate a sample enriched in nucleated cells. The cells ofinterest can subsequently be separated from the sample using methodsknown in the art.

When obtaining a sample from a subject (e.g., blood sample), the amountcan vary depending upon subject size and the condition being screened.In some embodiments, up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 mL of a sample is obtained. In some embodiments, 1-50, 2-40, 3-30,or 4-20 mL of sample is obtained. In some embodiments, more than 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 mL of a sample is obtained.

Nucleic Acids

Nucleic acids from samples that can be analyzed by the methods hereininclude: double-stranded DNA, single-stranded DNA, single-stranded DNAhairpins, DNA/RNA hybrids, RNA (e.g. mRNA or miRNA) and RNA hairpins.Examples of genetic analyses that can be performed on nucleic acidsinclude e.g., sequencing, SNP detection, STR detection, RNA expressionanalysis, and gene expression.

In some embodiments, less than 1 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg,50 pg, 100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng,50 ng, 100 ng, 200 ng, 500 ng, 1 ug, 5 ug, 10 ug, 20 ug, 30 ug, 40 ug,50 ug, 100 ug, 200 ug, 500 ug or 1 mg of nucleic acids are obtained fromthe sample for further genetic analysis. In some cases, about 1-5 pg,5-10 pg, 10-100 pg, 100 pg-1 ng, 1-5 ng, 5-10 ng, 10-100 ng, 100 ng-1 ugof nucleic acids are obtained from the sample for further geneticanalysis.

In some embodiments, the methods described herein are used to detectand/or quantified a target nucleic acid molecule. In some embodiments,the methods described herein are used to detect and/or quantifiedmultiple target nucleic acid molecules. The methods described herein cananalyzed at least 1; 2; 3; 4; 5; 10, 20; 50; 100; 200; 500; 1,000;2,000; 5,000; 10,000, 20,000; 50,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;2,000,000 or 3,000,000 different target nucleic acids.

In some embodiments, the methods described herein are used todistinguish between target nucleic acids that differ from anothernucleic acid by 1 nt. In some embodiments, the methods described hereinare used to distinguish between target nucleic acids that differ fromanother nucleic acid by 1 nt or more than 1, 2, 3, 5, 10, 15, 20, 21,22, 24, 25, 30 nt.

In some embodiments, the methods described herein are used to detectand/or quantify genomic DNA regions. In some embodiments, the methodsdescribed herein can discriminate and quantitate genomic DNA regions.The methods described herein can discriminate and quantitate at least 1;2; 3; 4; 5; 10, 20; 50; 100; 200; 500; 1,000; 2,000; 5,000; 10,000,20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 2,000,000 or 3,000,000 differentgenomic DNA regions. The methods described herein can discriminate andquantitate genomic DNA regions varying by 1 nt or more than 1, 2, 3, 5,10, 15, 20, 21, 22, 24, 25, 30 nt.

In some embodiments, the methods described herein are used to detectand/or quantify genomic DNA regions such as a region containing a DNApolymorphism. A polymorphism refers to the occurrence of two or moregenetically determined alternative sequences or alleles in a population.A polymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at afrequency of preferably greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude single nucleotide polymorphisms (SNP's), restriction fragmentlength polymorphisms (RFLP's), short tandem repeats (STRs), variablenumber of tandem repeats (VNTR's), hypervariable regions,minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. A polymorphism between two nucleic acids can occurnaturally, or be caused by exposure to or contact with chemicals,enzymes, or other agents, or exposure to agents that cause damage tonucleic acids, for example, ultraviolet radiation, mutagens orcarcinogens. In some embodiments, the methods described herein candiscriminate and quantitate a DNA region containing a DNA polymorphism.The methods described herein can discriminate and quantitate of at least1; 2; 3; 4; 5; 10, 20; 50; 100; 200; 500; 1,000; 2,000; 5,000; 10,000,20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 2,000,000 or 3,000,000 DNApolymorphism.

In some embodiments, the methods described herein can discriminate andquantitate at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 200; 500; 1,000;2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;2,000,000 or 3,000,000 different polymorphic markers.

In some embodiments, the methods described herein can discriminate andquantitate at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 200; 500; 1,000;2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;2,000,000 or 3,000,000 different SNPs.

In some embodiments, the methods described herein are used to detectand/or quantify gene expression. In some embodiments, the methodsdescribed herein provide high discriminative and quantitative analysisof multiples genes. The methods described herein can discriminate andquantitate the expression of at least 1, 2, 3, 4, 5, 10, 20, 50, 100,200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000,different target nucleic acids.

In some embodiments, the methods described herein are used to detectand/or quantify gene expression of genes with similar sequences. Themethods described herein can discriminate and quantitate the expressionof genes varying by 1 nt or more than 1, 2, 3, 4, 5, 10, 12, 15, 20, 21,22, 24, 25, 30 nt.

In some embodiments, the methods described herein are used to detectand/or quantify genomic DNA regions by mapping the region to the genomeof a species in the case where the transplant donor and the transplantrecipient are not from the same species (e.g., xenotransplants). In someembodiments, the methods described herein can discriminate andquantitate a DNA region from a species. The methods described herein candiscriminate and quantitate of at least 1; 2; 3; 4; 5; 10, 20; 50; 100;200; 500; 1,000; 2,000; 5,000; 10,000, 20,000; 50,000; 100,000; 200,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 2,000,000 or 3,000,000 DNA regions from a species.

In some embodiments, the methods described herein are used fordiagnosing or predicting transplant status or outcome (e.g. transplantrejection). In some embodiments, the methods described herein are usedto detect and/or quantify target nucleic acids to determine whether apatient or subject is displaying transplant tolerance. In someembodiments, the methods described herein are used to detect and/orquantify target nucleic acids for diagnosis or prediction of transplantrejection. In some embodiments, the methods described herein are used todetect and/or quantify target nucleic acids for determining animmunosuppressive regimen for a subject who has received a transplant,e.g., an allograft. In some embodiments, the methods described hereinare used to detect and/or quantify target nucleic acids to predicttransplant survival in a subject that have received a transplant. Theinvention provides methods of diagnosing or predicting whether atransplant in a transplant patient or subject will survive or be lost.In certain embodiments, the methods described herein are used to detectand/or quantify target nucleic acids to diagnose or predict the presenceof long-term graft survival. In some embodiments, the methods describedherein are used to detect and/or quantify target nucleic acids fordiagnosis or prediction of non-rejection based transplant injury.Examples of non-rejection based graft injury include, but are notlimited to, ischemic injury, virus infection, peri-operative ischemia,reperfusion injury, hypertension, physiological stress, injuries due toreactive oxygen species and injuries caused by pharmaceutical agents.

As used herein the term “diagnose” or “diagnosis” of a transplant statusor outcome includes predicting or diagnosing the transplant status oroutcome, determining predisposition to a transplant status or outcome,monitoring treatment of transplant patient, diagnosing a therapeuticresponse of transplant patient, and prognosis of transplant status oroutcome, transplant progression, and response to particular treatment.

Donor Organ Nucleic Acid Detection and Analysis

In some embodiments, the methods, devices, compositions and kits areused to establish a genotype for both the donor and the recipient beforetransplantation to enable the detection of donor-specific nucleic acidssuch as DNA or RNA in bodily fluids such as blood or urine from theorgan recipient after transplantation. This approach allows for areliable identification of sequences arising solely from the organtransplantation that can be made in a manner that is independent of thegenders of donor and recipient.

In some embodiments, a genetic fingerprint is generated for the donororgan. Both the donor and recipient will be genotyped prior totransplantation. Genotyping of transplant donors and transplantrecipients establishes a profile, using distinguishable markers, fordetecting donor nucleic acids (e.g. circulating cell-free nucleic acidor nucleic acids from circulating donor cells). In some embodiments, forxenotransplants, nucleic acids from the donors can be mapped to thegenome of the donor species.

Following transplantation, samples as described above can be drawn fromthe patient and analyzed for markers. The proportion of donor nucleicacids can be monitored over time and an increase in this proportion canbe used to determine transplant status or outcome (e.g. transplantrejection).

In some embodiments, genotyping comprises detection and quantitation ofnucleic acids from circulating transplant donor cells or circulatingcell-free nucleic acids. Examples of nucleic acids include, but are notlimited to double-stranded DNA, single-stranded DNA, single-stranded DNAhairpins, DNA/RNA hybrids, RNA (e.g. mRNA or miRNA) and RNA hairpins. Insome embodiments, the nucleic acid is DNA. In some embodiments, thenucleic acid is RNA. For instance, cell-free RNA is also present inhuman plasma (Tong, Y. K. Lo, Y. M., Clin Chim Acta, 363, 187-196(2006)) and cDNA sequencing of organ-specific transcripts providesanother option to detect donor-specific nucleic acids arising from cellsin the transplanted organ. In some embodiments, nucleic acids collectedfrom circulating cells in the blood are used.

In some embodiments, genotyping comprises detection and quantitation ofpolymorphic markers. Examples of polymorphic markers include singlenucleotide polymorphisms (SNP's), restriction fragment lengthpolymorphisms (RFLP's), variable number of tandem repeats (VNTR's),short tandem repeats (STRs), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. In someembodiments, genotyping comprises detection and quantitation of STRs. Insome embodiments, genotyping comprises detection and quantitation ofVNTRs.

In some embodiments, genotyping comprises detection and quantitation ofSNPs. Without intending to be limited to any theory, any donor andrecipient will vary at roughly three million SNP positions if fullygenotyped. Usable SNPs must be homozygous for the recipient and ideallyhomozygous for the donor as well. While the majority of these positionswill contain SNPs that are heterozygous for either the donor or therecipient, over 10% (or hundreds of thousands) will be homozygous forboth donor and recipient meaning a direct read of that SNP position candistinguish donor DNA from recipient DNA. For example, after genotypinga transplant donor and transplant recipient, using existing genotypingplatforms know in the art including the one described herein, one couldidentify approximately 1.2 million total variations between a transplantdonor and transplant recipient. Usable SNPs may comprise approximately500,000 heterozygous donor SNPs and approximately 160,000 homozygousdonor SNPs. Companies (such as Applied Biosystems, Inc.) currently offerboth standard and custom-designed TaqMan probe sets for SNP genotypingthat can in principle target any desired SNP position for a PCR-basedassay (Livak, K. L., Marmaro, J., Todd, J. A., Nature Genetics, 9,341-342 (1995); De La Vefa, F. M., Lazaruk, K. D., Rhodes, M. D., Wenz,M. H., Mutation Research, 573, 111-135 (2005)). With such a large poolof potential SNPs to choose from, a usable subset of existing or customprobes can be selected to serve as the probe set for any donor/recipientpair. In some embodiments, digital PCR or real-time PCR performed on thenucleic acids recovered from plasma or other biological samples willdirectly quantitate the percentage of donor-specific species seen in thesample. In some embodiments, sequencing performed on the nucleic acidrecovered from plasma or other biological samples will directlyquantitate the percentage of donor-specific species seen in the sample.In some embodiments, arrays can be used on the nucleic acids recoveredfrom plasma or other biological samples to directly quantitate thepercentage of donor-specific species seen in the sample.

Due to the low number of expected reads for any individual nucleic acid(e.g. SNP) in patient samples, some preamplification of the samplematerial may be required before analysis to increase signal levels, butusing either preamplification, sampling more target nucleic acidpositions (e.g. SNP positions), or both, will provide a reliableread-out of the transplant donor nucleic acid fraction.Pre-amplification can be preformed using any suitable method known inthe art such as multiple displacement amplification (MDA) (Gonzalez etal. Envircon Microbiol; 7(7); 1024-8 (2005)) or amplification with outerprimers in a nested PCR approach. This permits detection and analysis ofdonor nucleic acids even if the total amount of donor nucleic acid inthe sample (e.g. blood from transplant patient) is only up to 1 μg, 500ng, 200 ng, 100 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, 1 ng, 500pg, 200 pg, 100 pg, 50 pg, 40 pg, 30 pg, 20 p, 10 pg, 5 pg, or 1 pg orbetween 1 5 μg, 5 10 μg, or 10 50 μg.

a. PCR

Genotyping donor and recipient nucleic acids, and/or detection,identification and/or quantitation of the donor-specific nucleic acidsafter transplantation (e.g. polymorphic markers such as SNPs) can beperformed by PCR. Examples of PCR techniques that can be used to detect,identify and/or quantitate the donor-specific nucleic acids include, butare not limited, to quantitative PCR, quantitative fluorescent PCR(QF-PCR), multiplex fluorescent PCR (MF-PCR), real time PCR (RT-PCR),single cell PCR, restriction fragment length polymorphism PCR(PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, in situpolonony PCR, in situ rolling circle amplification (RCA), bridge PCR,picotiter PCR and emulsion PCR. Other suitable amplification methodsinclude the ligase chain reaction (LCR), transcription amplification,self-sustained sequence replication, selective amplification of targetpolynucleotide sequences, consensus sequence primed polymerase chainreaction (CP-PCR), arbitrarily primed polymerase chain reaction(AP-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR) and nucleicacid based sequence amplification (NABSA). Other amplification methodsthat may be used to amplify specific polymorphic loci include thosedescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and6,582,938. In some embodiments, Detection, identification and/orquantitation of the donor-specific nucleic acids (e.g. polymorphicmarkers such as SNPs) is performed by real-time PCR.

In some embodiments, digital PCR or real time PCR to quantitate thepresence of specific polymorphisms that have already been identified inthe initial genotyping step pre-transplantation. Compared with thequantitative PCR techniques used in some of the earlier cited work,digital PCR is a much more accurate and reliable method to quantitatenucleic acid species including rare nucleic acid species, and does notrequire a specific gender relationship between donor and recipient.(Warren, L., Bryder, D., Weissman, I. L., Quake, S. R., Proc Natl AcadSci, 103, 17807-17812 (2006)). In some embodiments, digital PCR orreal-time PCR assays can be used to quantitate the fraction of donor DNAin a transplant patient using probes targeted to several SNPs.

b. Sequencing

Genotyping donor and recipient nucleic acids, and/or detection,identification and/or quantitation of the donor-specific nucleic acidsafter transplantation (e.g. polymorphic markers such as SNPs) can beperformed by sequencing such as whole genome sequencing or exomesequencing. Sequencing can be accomplished through classic Sangersequencing methods which are well known in the art. Sequence can also beaccomplished using high-throughput systems some of which allow detectionof a sequenced nucleotide immediately after or upon its incorporationinto a growing strand, i.e., detection of sequence in red time orsubstantially real time. In some cases, high throughput sequencinggenerates at least 1,000, at least 5,000, at least 10,000, at least20,000, at least 30,000, at least 40,000, at least 50,000, at least100,000 or at least 500,000 sequence reads per hour; with each readbeing at least 50, at least 60, at least 70, at least 80, at least 90,at least 100, at least 120 or at least 150 bases per read. Sequencingcan be preformed using nucleic acids described herein such as genomicDNA, cDNA derived from RNA transcripts or RNA as a template.

In some embodiments, high-throughput sequencing involves the use oftechnology available by Helicos BioSciences Corporation (Cambridge,Mass.) such as the Single Molecule Sequencing by Synthesis (SMSS)method. SMSS is unique because it allows for sequencing the entire humangenome with no pre amplification step needed. Thus, distortion andnonlinearity in the measurement of nucleic acids are reduced. Thissequencing method also allows for detection of a SNP nucleotide in asequence in substantially real time or real time. Finally, as mentionedabove, SMSS is powerful because, like the MIP technology, it does notrequire a pre amplification step prior to hybridization. In fact, SMSSdoes not require any amplification. SMSS is described in part in USPublication Application Nos. 2006002471 I; 20060024678; 20060012793;20060012784; and 20050100932.

In some embodiments, high-throughput sequencing involves the use oftechnology available by 454 Lifesciences, Inc. (Branford, Conn.) such asthe Pico Titer Plate device which includes a fiber optic plate thattransmits chemiluminescent signal generated by the sequencing reactionto be recorded by a CCD camera in the instrument. This use of fiberoptics allows for the detection of a minimum of 20 million base pairs in4.5 hours.

Methods for using bead amplification followed by fiber optics detectionare described in Marguiles, M., et al. “Genome sequencing inmicrofabricated high-density picoliter reactors”, Nature, doi:10.1038/nature03959; and well as in US Publication Application Nos.200200 12930; 20030058629; 20030 1001 02; 20030 148344; 20040248 161;200500795 10,20050 124022; and 20060078909.

In some embodiments, high-throughput sequencing is performed usingClonal Single Molecule Array (Solexa, Inc.) or sequencing-by-synthesis(SBS) utilizing reversible terminator chemistry. These technologies aredescribed in part in U.S. Pat. Nos. 6,969,488; 6,897,023; 6,833,246;6,787,308; and US Publication Application Nos. 200401061 30;20030064398; 20030022207; and Constans, A., The Scientist 2003,17(13):36.

In some embodiments of this aspect, high-throughput sequencing of RNA orDNA can take place using AnyDot.chips (Genovoxx, Germany), which allowsfor the monitoring of biological processes (e.g., miRNA expression orallele variability (SNP detection). In particular, the AnyDot-chipsallow for 10×-50× enhancement of nucleotide fluorescence signaldetection. AnyDot.chips and methods for using them are described in partin International Publication Application Nos. WO 02088382, WO 03020968,WO 0303 1947, WO 2005044836, PCTEP 05105657, PCMEP 05105655; and GermanPatent Application Nos. DE 101 49 786, DE 102 14 395, DE 103 56 837, DE10 2004 009 704, DE 10 2004 025 696, DE 10 2004 025 746, DE 10 2004 025694, DE 10 2004 025 695, DE 10 2004 025 744, DE 10 2004 025 745, and DE10 2005 012 301.

Other high-throughput sequencing systems include those disclosed inVenter, J., et al. Science 16 Feb. 2001; Adams, M. et al, Science 24Mar. 2000; and M. J, Levene, et al. Science 299:682-686, January 2003;as well as US Publication Application No. 20030044781 and 2006/0078937.Overall such system involve sequencing a target nucleic acid moleculehaving a plurality of bases by the temporal addition of bases via apolymerization reaction that is measured on a molecule of nucleic acid,i e., the activity of a nucleic acid polymerizing enzyme on the templatenucleic acid molecule to be sequenced is followed in real time. Sequencecan then be deduced by identifying which base is being incorporated intothe growing complementary strand of the target nucleic acid by thecatalytic activity of the nucleic acid polymerizing enzyme at each stepin the sequence of base additions. A polymerase on the target nucleicacid molecule complex is provided in a position suitable to move alongthe target nucleic acid molecule and extend the oligonucleotide primerat an active site. A plurality of labeled types of nucleotide analogsare provided proximate to the active site, with each distinguishablytype of nucleotide analog being complementary to a different nucleotidein the target nucleic acid sequence. The growing nucleic acid strand isextended by using the polymerase to add a nucleotide analog to thenucleic acid strand at the active site, where the nucleotide analogbeing added is complementary to the nucleotide of the target nucleicacid at the active site. The nucleotide analog added to theoligonucleotide primer as a result of the polymerizing step isidentified. The steps of providing labeled nucleotide analogs,polymerizing the growing nucleic acid strand, and identifying the addednucleotide analog are repeated so that the nucleic acid strand isfurther extended and the sequence of the target nucleic acid isdetermined.

In some embodiments, shotgun sequencing is performed. In shotgunsequencing, DNA is broken up randomly into numerous small segments,which are sequenced using the chain termination method to obtain reads.Multiple overlapping reads for the target DNA are obtained by performingseveral rounds of this fragmentation and sequencing. Computer programsthen use the overlapping ends of different reads to assemble them into acontinuous sequence

In some embodiments, the invention provides methods for detection andquantitation of SNPs using sequencing. In this case, one can estimatethe sensitivity of detection. There are two components to sensitivity:(i) the number of molecules analyzed (depth of sequencing) and (ii) theerror rate of the sequencing process. Regarding the depth of sequencing,a frequent estimate for the variation between individuals is that aboutone base per thousand differs. Currently, sequencers such as theIllumina Genome Analyzer have read lengths exceeding 36 base pairs.Without intending to be limited to any theory or specific embodiment,this means that roughly one in 30 molecules analyzed will have apotential SNP. While the fraction of donor DNA in the recipient blood iscurrently not well determined and will depend on organ type, one cantake 1% as a baseline estimate based on the literature and applicantsown studies with heart transplant patients. At this fraction of donorDNA, approximately one in 3,000 molecules analyzed will be from thedonor and informative about donor genotype. On the Genome Analyzer onecan obtain about 10 million molecules per analysis channel and there are8 analysis channels per instrument run. Therefore, if one sample isloaded per channel, one should be able to detect about 3,000 moleculesthat can be identified as from the donor in origin, more than enough tomake a precise determination of the fraction of donor DNA using theabove parameters. If one wants to establish a lower limit of sensitivityfor this method by requiring at least 100 donor molecules to bedetected, then it should have a sensitivity capable of detecting donormolecules when the donor fraction is as low as 0.03%. Higher sensitivitycan be achieved simply by sequencing more molecules, i.e. using morechannels.

The sequencing error rate also affects the sensitivity of thistechnique. For an average error rate of ε, the chance of a single SNPbeing accidentally identified as of donor origin as a result of amis-read is roughly ε/3. For each individual read, this establishes alower limit of sensitivity of one's ability to determine whether theread is due to donor or recipient. Typical sequencing error rates forbase substitutions vary between platforms, but are between 0.5-1.5%.This places a potential limit on sensitivity of 0.16 to 0.50%. However,it is possible to systematically lower the sequencing error rate byresequencing the sample template multiple times, as has beendemonstrated by Helicos BioSciences (Harris, T. D., et al., Science,320, 106-109 (2008)). A single application of resequencing would reducethe expected error rate of donor SNP detection to ε²/9 or less than0.003%.

FIG. 5 shows in one embodiments of the inventions a general strategy formonitor all patients, (i.e., not just female patients receiving maleorgans), to determine a transplants status or outcome. Genotyping ofdonor and recipient can establish a single nucleotide polymorphism (SNP)profile for detecting donor DNA. Shotgun sequencing of cell-free DNA inplasma, with analysis of observed unique SNPs, allows quantitation of %donor DNA. While any single SNP may be difficult to detect with solittle DNA in plasma, with hundred of thousands or more signals toconsider, high sensitivity should be possible.

c. Arrays

Genotyping donor and recipient nucleic acids, and/or detection,identification and/or quantitation of the donor-specific nucleic acidsafter transplantation (e.g. polymorphic markers such as SNPs) can beperformed using arrays (e.g. SNPs arrays). Results can be visualizedusing a scanner that enables the viewing of intensity of data collectedand software to detect and quantify nucleic acid. Such methods aredisclosed in part U.S. Pat. No. 6,505,125. Another method contemplatedby the present invention to detect and quantify nucleic acids involvesthe use of bead as is commercially available by Illumina, Inc. (SanDiego) and as described in U.S. Pat. Nos. 7,035,740; 7,033,754;7,025,935, 6,998,274; 6,942,968; 6,913,884; 6,890,764; 6,890,741;6,858,394; 6,812,005; 6,770,441; 6,620,584; G,544,732; 6,429,027;6,396,995; 6,355,431 and US Publication Application Nos. 20060019258;0050266432; 20050244870; 20050216207; 20050181394; 20050164246;20040224353; 20040185482; 20030198573; 20030175773; 20030003490;20020187515; and 20020177141; and in B. E. Stranger, et al., PublicLibrary of Science-Genetics, I (6), December 2005; Jingli Cai, el al.,Stem Cells, published online Nov. 17, 2005; C. M. Schwartz, et al., StemCells and Development, f 4, 517-534, 2005; Barnes, M., J. el al.,Nucleic Acids Research, 33 (1 81, 5914-5923, October 2005; and BibikovaM, et al. Clinical Chemistry, Volume 50, No. 12, 2384-2386, December2004. Additional description for preparing RNA for bead arrays isdescribed in Kacharmina J E, et al., Methods Enzymol 303: 3-18, 1999;Pabon C, et al., Biotechniques 3 1(4): 8769, 2001; Van Gelder R N, eta]., Proc Natl Acad Sci USA 87: 1663-7 (1990); and Murray, S S. BMCGenetics B(SupplI):SX5 (2005).

When analyzing SNP according to the methods described herein, thetransplant donor and/or recipient nucleic acids can be labeled andhybridized with a DNA microarray (e.g., 100K Set Array or other array).Results can be visualized using a scanner that enables the viewing ofintensity of data collected and software “calls” the SNP present at eachof the positions analyzed. Computer implemented methods for determininggenotype using data h m mapping arrays are disclosed, for example, inLiu, et al., Bioinformatics 19:2397-2403, 2003; and Di et al.,Bioinformatics 21: 1958-63, 2005. Computer implemented methods forlinkage analysis using mapping array data are disclosed, for example, inRuschendorf and Nusnberg, Bioinfonnatics 21:2123-5, 2005; and Leykin eta]., BMC Genet. 6:7, 2005; and in U.S. Pat. No. 5,733,729.

In some embodiments of this aspect, genotyping microarrays that are usedto detect SNPs can be used in combination with molecular inversionprobes (MIPS) as described in Hardenbol et al., Genome Res.15(2):269-275, 2005, Hardenbol, P. et al. Nature Biotechnology 2 1 (6),673-8, 2003; Faham M, et al. Hum Mol Genet. August 1; 10(16): 1657-64,2001: Maneesh Jain, Ph.D., et al. Genetic Engineering News V24: No. 18,2004; and Fakhrai-Rad H, el al. Genome Res. July; 14(7):1404-12, 2004;and in U.S. Pat. No. 5,858,412. Universal tag arrays and reagent kitsfor performing such locus specific genotyping using panels of customMIPs are available from Affymetrix and ParAllele. MIP technologyinvolves the use enzymological reactions that can score up to 10,000:20,000, 50,000; 100,000; 200,000; 500,000; 1,000,000; 2,000,000 or5,000,000 SNPs (target nucleic acids) in a single assay. Theenzymological reactions are insensitive to crossreactivity amongmultiple probe molecules and there is no need for pre-amplificationprior to hybridization of the probe with the genomic DNA. In any of theembodiments, the target nucleic acid(s) or SNPs can be obtained from asingle cell.

Another method contemplated by the present invention to detect targetnucleic acids involves the use of bead arrays (e.g., such as onecommercially available by Illumina, Inc.) as described in U.S. Pat. Nos.7,040,959; 7,035,740; 7,033,754; 7,025,935, 6,998,274; 6,942,968;6,913,884; 6,890,764; 6,890,741; 6,858,394; 6,846,460; 6,812,005;6,770,441; 6,663,832; 5,520,584; 6,544,732; 6,429,027; 6,396,995;6,355,431 m d US Publication Application Nos. 20060019258; 20050266432;20050244870; 20050216207; 20050181394; 20050164246; 20040224353;20040185482; 20030198573; 200301 75773; 20030003490; 200201 8751 5; and20020177141; as well as Shen, R., et al. Mutation Research 573 70-82(2005).

d. Other Techniques

In some of the embodiment herein, nucleic acids are quantified. Methodsfor quantifying nucleic acids are known in the art and include, but arenot limited to, gas chromatography, supercritical fluid chromatography,liquid chromatography (including partition chromatography, adsorptionchromatography, ion exchange chromatography, size exclusionchromatography, thin-layer chromatography, and affinity chromatography),electrophoresis (including capillary electrophoresis, capillary zoneelectrophoresis, capillary isoelectric focusing, capillaryelectrochromatography, micellar electrokinetic capillary chromatography,isotachophoresis, transient isotachophoresis and capillary gelelectrophoresis), comparative genomic hybridization (CGH), microarrays,bead arrays, and high-throughput genotyping such as with the use ofmolecular inversion probe (MIP).

Another method contemplated by the present invention to detect and/orquantify target nucleic acids involves the use of nanoreporters asdescribed in U.S. Pat. No. 7,473,767 entitled “Methods for detection andquantification of analytes in complex mixtures”, US patent publicationno. 2007/0166708 entitled “Methods for detection and quantification ofanalytes in complex mixtures”, U.S. application Ser. No. 11/645,270entitled “Compositions comprising oriented, immobilized macromoleculesand methods for their preparation”, PCT application no U.S. Ser. No.06/049,274 entitled “Nanoreporters and methods of manufacturing and usethereof”,

Quantification of target nucleic acid can be used to determine thepercentage of donor nucleic acids such as DNA.

e. Labels

Detection and/or quantification of target nucleic acids can be doneusing fluorescent dyes known in the art. Fluorescent dyes may typicallybe divided into families, such as fluorescein and its derivatives;rhodamine and its derivatives; cyanine and its derivatives; coumarin andits derivatives; Cascade Blue™ and its derivatives; Lucifer Yellow andits derivatives; BODIPY and its derivatives; and the like. Exemplaryfluorophores include indocarbocyanine (C3), indodicarbocyanine (C5),Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488,Alexa Fluor®-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green,BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM),phycoerythrin, rhodamine, dichlororhodamine (dRhodamine™), carboxytetramethylrhodamine (TAMRA™), carboxy-X-rhodamine (ROX™), LIZ™, VIC™,NED™, PET™, SYBR, PicoGreen, RiboGreen, and the like. Descriptions offluorophores and their use, can be found in, among other places, R.Haugland, Handbook of Fluorescent Probes and Research Products, 9.sup.thed. (2002), Molecular Probes, Eugene, Oreg.; M. Schena, MicroarrayAnalysis (2003), John Wiley & Sons, Hoboken, N.J.; Synthetic MedicinalChemistry 2003/2004 Catalog, Berry and Associates, Ann Arbor, Mich.; G.Hermanson, Bioconjugate Techniques, Academic Press (1996); and GlenResearch 2002 Catalog, Sterling, Va. Near-infrared dyes are expresslywithin the intended meaning of the terms fluorophore and fluorescentreporter group.

In another aspect of the invention, a branched-DNA (bDNA) approach isused to increase the detection sensitivity. In some embodiments, bDNAapproach is applied to an array detection assay. The array detectionassay can be any array assay known in the art, including the arrayassays described herein. bDNA approach amplifies the signals through abranched DNA that are attached by tens or hundreds of alkalinephosphatase molecules. Thus, the signals are significantly amplifiedwhile the fidelity of the original nucleic acid target abundance ismaintained.

Methods

In one aspect the invention provides methods for the diagnosis orprediction of transplant status or outcome in a subject who has receiveda transplant. The transplant status or outcome may comprise rejection,tolerance, non-rejection based transplant injury, transplant function,transplant survival, chronic transplant injury, or titer pharmacologicalimmunosuppression. Examples of non-rejection based allograft injuryinclude, but are not limited to, ischemic injury, virus infection,peri-operative ischemia, reperfusion injury, hypertension, physiologicalstress, injuries due to reactive oxygen species and injuries caused bypharmaceutical agents. The transplant status or outcome may comprisevascular complications or neoplastic involvement of the transplantedorgan.

In some embodiments, the invention provides methods of diagnosing orpredicting transplant status or outcome comprising the steps of: (i)providing a sample from a subject who has received a transplant from adonor; (ii) determining the presence or absence of one or more nucleicacids from the donor transplant, wherein the one or more nucleic acidsfrom the donor are identified based on a predetermined marker profile;and (iii) diagnosing or predicting transplant status or outcome based onthe presence or absence of the one or more nucleic acids from saiddonor.

In some embodiments, the methods of the invention are used to establisha genotype for both the donor and the recipient before transplantation.In some embodiments, the genotyping of both the donor and the recipientbefore transplantation enables the detection of donor-specific nucleicacids such as DNA or RNA in bodily fluids as described herein (e.g.,blood or urine) from the organ recipient after transplantation. In someembodiments a marker profile for the donor is determined based on thegenotyping of the transplant donor. In some embodiments, a markerprofile is determined for the transplant recipient based on thegenotyping of the transplant recipient. In some embodiments, a markerprofile is established by selecting markers that are distinguishablebetween the transplant donor and the subject receiving the transplant.This approach allows for a reliable identification of nucleic acidsarising solely from the organ transplantation that can be made in amanner that is independent of the genders of donor and recipient.

Genotyping of the transplant donor and/or the transplant recipient maybe performed by any suitable method known in the art including thosedescribed herein such as sequencing, nucleic acid array or PCR. In someembodiments, genotyping of the transplant donor and/or the transplantrecipient is performed by shotgun sequencing. In some embodiments,genotyping of the transplant donor and/or the transplant recipient isperformed using a DNA array. In some embodiments, genotyping of thetransplant donor and/or the transplant recipient is performed using apolymorphism array such as a SNP array.

In some embodiments, the marker profile is a polymorphic marker profile.Polymorphic marker profile may comprise one or more single nucleotidepolymorphisms (SNP's), one or more restriction fragment lengthpolymorphisms (RFLP's), one or more short tandem repeats (STRs), one ormore variable number of tandem repeats (VNTR's), one or morehypervariable regions, one or more minisatellites, one or moredinucleotide repeats, one or more trinucleotide repeats, one or moretetranucleotide repeats, one or more simple sequence repeats, or one ormore insertion elements. In some embodiments, the marker profilecomprises at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 200; 500; 1,000;2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;2,000,000 or 3,000,000 different polymorphic markers.

In some embodiments, the polymorphic marker profile comprises one ormore SNPs. In some embodiments, the marker profile comprises at least 1;2; 3; 4; 5; 10; 20; 50; 100; 200; 500; 1,000; 2,000; 5,000; 10,000;20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 2,000,000 or 3,000,000 differentSNPs.

Following transplantation, samples as described above can be drawn fromthe patient and analyzed for the presence or absence of one or morenucleic acids from the transplant donor. In some embodiments, the sampleis blood, plasma, serum or urine. The proportion and/or amount of donornucleic acids can be monitored over time and an increase in thisproportion can be used to determine transplant status or outcome (e.g.transplant rejection).

The presence or absence of one or more nucleic acids from the transplantdonor in the transplant recipient may be determined by any suitablemethod known in the art including those described herein such assequencing, nucleic acid arrays or PCR. In some embodiments, thepresence or absence of one or more nucleic acids from the transplantdonor in the transplant recipient is determined by shotgun sequencing.In some embodiments, the presence or absence of one or more nucleicacids from the transplant donor in the transplant recipient isdetermined using a DNA array. In some embodiments, the presence orabsence of one or more nucleic acids from the transplant donor in thetransplant recipient is determined using a polymorphism array such as aSNP array.

In some embodiments, where the transplant is a xenotransplant,detection, identification and/or quantitation of the donor-specificmarkers can be performed by mapping one or more nucleic acids (e.g.,DNA) to the genome of the specie use to determine whether the one ormore nucleic acids come from the transplant donor. Polymorphic markersas described above can also be used where the transplant is axenotransplant.

In some embodiments, the presence or absence of circulating DNA or RNAfrom a transplant donor in a transplant recipient is used to determinethe transplant status or outcome. The DNA can be double-stranded DNA,single-stranded DNA, single-stranded DNA hairpins, or cDNA. The RNA canbe single stranded RNA or RNA hairpins. In some embodiments, thepresence or absence of circulating DNA/RNA hybrids from a transplantdonor in a transplant recipient is used to determine the transplantstatus or outcome. In some embodiments, the presence or absence ofcirculating mRNA from a transplant donor in a transplant recipient isused to determine the transplant status or outcome. In some embodiments,the presence or absence of circulating DNA from a transplant donor in atransplant recipient is used to determine the transplant status oroutcome. In some embodiments, cDNA is used to determine the transplantstatus or outcome. The DNA or RNA can be obtained from circulating donorcells. Alternative, the DNA or RNA can be circulating cell-free DNA orcirculating cell-free RNA

In any of the embodiments described herein, the transplant graft maybeany solid organ and skin transplant. Examples of transplants, whosetransplant status or outcome could be determined by the methodsdescribed herein, include but are not limited to, kidney transplant,heart transplant, liver transplant, pancreas transplant, lungtransplant, intestine transplant and skin transplant.

In some embodiments, the invention provides methods of determiningwhether a patient or subject is displaying transplant tolerance. In someembodiments the invention provides methods for diagnosis or predictionof transplant rejection. The term “transplant rejection” encompassesboth acute and chronic transplant rejection. In some embodiments, theinvention further includes methods for determining an immunosuppressiveregimen for a subject who has received a transplant, e.g., an allograft.In some embodiments, the invention further includes methods fordetermining the effectiveness of an immunosuppressive regimen for asubject who has received a transplant. Certain embodiments of theinvention provide methods of predicting transplant survival in a subjectthat has received a transplant. The invention provides methods ofdiagnosing or predicting whether a transplant in a transplant patient orsubject will survive or be lost. In certain embodiments, the inventionprovides methods of diagnosing or predicting the presence of long-termgraft survival. In some embodiments, the invention provides methods fordiagnosis or prediction of non-rejection based transplant injury.Examples of non-rejection based graft injury include, but are notlimited to, ischemic injury, virus infection, peri-operative ischemia,reperfusion injury, hypertension, physiological stress, injuries due toreactive oxygen species and injuries caused by pharmaceutical agents. Insome embodiments, the invention provides methods for diagnosis orprediction of vascular complications or neoplastic involvement of thetransplanted organ.

In some embodiments, the amount of one or more nucleic acids from thetransplant donor in a sample from the transplant recipient is used todetermine the transplant status or outcome. Thus, in some embodiments,the methods of the invention further comprise quantitating the one ormore nucleic acids from the transplant donor. In some embodiments, theamount of one or more nucleic acids from the donor sample is determinedas a percentage of total the nucleic acids in the sample. In someembodiments, the amount of one or more nucleic acids from the donorsample is determined as a ratio of the total nucleic acids in thesample. In some embodiments, the amount of one or more nucleic acidsfrom the donor sample is determined as a ratio or percentage compared toone or more reference nucleic acids in the sample. For instance, theamount of one or more nucleic acids from the transplant donor can bedetermined to be 10% of the total nucleic acids in the sample.Alternatively, the amount of one or more nucleic acids from thetransplant donor can be at a ratio of 1:10 compared to total nucleicacids in the sample. Further, the amount of one or more nucleic acidsfrom the transplant donor can be determined to be 10% or at a ratio of1:10 of a reference gene such a β-globin. In some embodiments, theamount of one or more nucleic acids from the transplant donor can bedetermined as a concentration. For example, the amount of one or morenucleic acids from the donor sample can be determined to be 1 ug/mL.

In some embodiments, the amount of one or more nucleic acids from thetransplant donor above a predetermined threshold value is indicative ofa transplant status or outcome. For example, the normative values forclinically stable post-transplantation patients with no evidence ofgraft rejection or other pathologies can be determined. An increase inthe amount of one or more nucleic acids from the transplant donor abovethe normative values for clinically stable post-transplantation patientscould indicate a change in transplant status or outcome such astransplant rejection or transplant injury. On the other hand, an amountof one or more nucleic acids from the transplant donor below or at thenormative values for clinically stable post-transplantation patientscould indicate graft tolerance or graft survival.

In some embodiments, different predetermined threshold values areindicative of different transplant outcomes or status. For example, asdiscussed above, an increase in the amount of one or more nucleic acidsfrom the transplant donor above the normative values for clinicallystable post-transplantation patients could indicate a change intransplant status or outcome such as transplant rejection or transplantinjury. However, an increase in the amount of one or more nucleic acidsfrom the transplant donor above the normative values for clinicallystable post-transplantation patients but below a predetermined thresholdlevel could indicate a less serious condition such as a viral infectionrather than transplant rejection. An increase in the amount of one ormore nucleic acids from the transplant donor above a higher thresholdcould indicate transplant rejection.

In some embodiments, temporal differences in the amount of said one ormore nucleic acids from the transplant donor are indicative of atransplant status or outcome. For instance, a transplant patient can bemonitored over time to determine the amount of one or more nucleic acidsfrom the transplant donor. A temporary increase in the amount of one ormore nucleic acids from the transplant donor, which subsequently returnto normal values, might indicate a less serious condition rather thantransplant rejection. On the other hand, a sustained increase in theamount one or more nucleic acids from the transplant donor mightindicate a serious condition such as transplant rejection.

In some embodiments, temporal differences in the amount of said one ormore nucleic acids from the transplant donor can be used to monitoreffectiveness of an immunosuppressant treatment or to select animmunosuppressant treatment. For instance, the amount of one or morenucleic acids from the transplant donor can be determined before andafter an immunosuppressant treatment. A decrease in the one or morenucleic acids from the transplant donor after treatment may indicatethat the treatment was successful in preventing transplant rejection.Additionally, the amount of one or more nucleic acids from thetransplant donor can be used to choose between immunosuppressanttreatments, for examples, immunosuppressant treatments of differentstrengths. For example, a higher amount in one or more nucleic acidsfrom the transplant donor may indicate that there is a need of a verypotent immunosuppressant, whereas a lower amount in one or more nucleicacids from the transplant donor may indicate that a less potentimmunosuppressant may be used.

The invention provides methods that sensitive and specific. In someembodiments, the methods described herein for diagnosing or predictingtransplant status or outcome have at least 56%, 60%, 70%, 80%, 90%, 95%or 100% sensitivity. In some embodiments, the methods described hereinhave at least 56% sensitivity. In some embodiments, the methodsdescribed herein have at least 78% sensitivity. In some embodiments, themethods described herein have a specificity of about 70% to about 100%.In some embodiments, the methods described herein have a specificity ofabout 80% to about 100%. In some embodiments, the methods describedherein have a specificity of about 90% to about 100%. In someembodiments, the methods described herein have a specificity of about100%.

Also provided herein are methods for screening and identifying markersrecognizing a donor nucleic acid that can be useful in the methodsdescribed herein, e.g. diagnosing or predicting transplant status oroutcome. In some embodiments, the donor nucleic acid is cell-free DNA orDNA isolated from circulating donor cells.

Donor nucleic acid can be identified by the methods described hereinincluding the methods described in the Examples. After identifyingthese, then one could look at the donor nucleic acids and examine themfor their correlation with transplant status and outcomes such aschronic graft injury, rejection, and tolerance. In some embodiments, thelongitudinal change of donor nucleic acids is studied. If clinicallysignificant, these levels could be followed to titer pharmacologicalimmunosuppression, or could be studied as a target for depletion.

Kits

Also provided are reagents and kits thereof for practicing one or moreof the above-described methods. The subject reagents and kits thereofmay vary greatly. Reagents of interest include reagents specificallydesigned for use in production of the above-described: (i) genotyping ofa transplant donor and a transplant recipient; (ii) identification ofmarker profiles; and (ii) detection and/or quantitation of one or morenucleic acids from a transplant donor in a sample obtained from atransplant recipient.

One type of such reagents are one or more probes or an array of probesto genotype and/or to detect and/or to quantitate one or more nucleicacids. A variety of different array formats are known in the art, with awide variety of different probe structures, substrate compositions andattachment technologies.

The kits of the subject invention may include the above-describedarrays. Such kits may additionally comprise one or more therapeuticagents. The kit may further comprise a software package for dataanalysis, which may include reference profiles for comparison with thetest profile.

The kits may comprise reagents such as buffers, and H₂0. The kits maycomprise reagents necessary to perform nucleic acid extraction and/ornucleic acid detection using the methods described herein such as PCRand sequencing.

Such kits may also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such kits may alsoinclude instructions to access a database. Such information may be basedon the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like. Kits may also, in some embodiments,be marketed directly to the consumer.

Computer Program

Any of the methods above can be performed by a computer program productthat comprises a computer executable logic that is recorded on acomputer readable medium. For example, the computer program can executesome or all of the following functions: (i) controlling isolation ofnucleic acids from a sample, (ii) pre-amplifying nucleic acids from thesample, (iii) amplifying, sequencing or arraying specific polymorphicregions in the sample, (iv) identifying and quantifying a marker profilein the sample, (v) comparing data on marker profile detected from thesample with a predetermined threshold, (vi) determining a transplantstatus or outcome, (vi) declaring normal or abnormal transplant statusor outcome. In particular, the computer executable logic can analyzedata on the detection and quantity of polymorphism(s) (e.g. SNPs).

The computer executable logic can work in any computer that may be anyof a variety of types of general-purpose computers such as a personalcomputer, network server, workstation, or other computer platform now orlater developed. In some embodiments, a computer program product isdescribed comprising a computer usable medium having the computerexecutable logic (computer software program, including program code)stored therein. The computer executable logic can be executed by aprocessor, causing the processor to perform functions described herein.In other embodiments, some functions are implemented primarily inhardware using, for example, a hardware state machine. Implementation ofthe hardware state machine so as to perform the functions describedherein will be apparent to those skilled in the relevant arts.

The program can provide a method of evaluating a transplant status oroutcome in a transplant recipient by accessing data that reflects thegenotyping of the transplant donor and the transplant patient, and/orthe presence or absence of one or more nucleic acids from the transplantdonor in the circulation of the transplant patient post-transplantation.

In one embodiment, the computer executing the computer logic of theinvention may also include a digital input device such as a scanner. Thedigital input device can provide information on a nucleic acid, e.g.,polymorphism levels/quantity. For example, a scanner of this inventioncan provide an image of the polymorphism (e.g., SNPs) according tomethod herein. For instance, a scanner can provide an image by detectingfluorescent, radioactive, or other emission; by detecting transmitted,reflected, or scattered radiation; by detecting electromagneticproperties or other characteristics; or by other techniques. The datadetected is typically stored in a memory device in the form of a datafile. In one embodiment, a scanner may identify one or more labeledtargets. For instance, a first DNA polymorphism may be labeled with afirst dye that fluoresces at a particular characteristic frequency, ornarrow band of frequencies, in response to an excitation source of aparticular frequency. A second DNA polymorphism may be labeled with asecond dye that fluoresces at a different characteristic frequency. Theexcitation sources for the second dye may, but need not, have adifferent excitation frequency than the source that excites the firstdye, e.g., the excitation sources could be the same, or different,lasers.

In some embodiments, the invention provides a computer readable mediumcomprising a set of instructions recorded thereon to cause a computer toperform the steps of (i) receiving data from one or more nucleic acidsdetected in a sample from a subject who has received transplant from adonor, wherein said one or more nucleic acids are nucleic acids fromsaid donor transplant, and wherein said one or more nucleic acids fromsaid donor are identified based on a predetermined marker profile; and(ii) diagnosing or predicting transplant status or outcome based on thepresence or absence of the one or more nucleic acids.

EXAMPLES Example 1: Detection of Donor DNA in Organ TransplantRecipients

Using digital PCR as described before (Warren, L., Bryder, D., Weissman,I. L., Quake, S. R., Proc Natl Acad Sci, 103, 17807-17812 (2006); Fan,H. C. Quake, S. R., Anal Chem, 79, 7576-7579 (2007)), the amount ofchromosome Y and chromosome 1 markers were quantitated for femalepatients receiving either male or female hearts in plasma samples takenat the same time that an endomyocardial biopsy determined a grade 3A or3B rejection episode.

While blood transfusions/male child birth are known mechanisms to havedetectable cY signature in a female patient, FIG. 2 shows that theoverall levels of cY are uniformly higher for patients receiving heartsfrom male donors. No significant chromosome Y signal from four controlfemale-to-female transplant patients was detected. On the other hand,1.5-8% total genomic fraction for chromosome Y signals was observed atthe rejection time points for three male-to-female transplant patientsacross four rejection episodes.

Levels of chromosome Y in plasma were monitored at several time pointsfollowing transplantation for some of these patients, and compared withbiopsy time points for organ rejection. For patient 6, a 3A graderejection was detected after biopsy 21 months after transplant. Thelevel of chromosome Y detected in plasma was neglible in plasma at threemonths prior to rejection, but increased >10-fold to 2% of total genomicfraction at the time a biopsy determined rejection. The highest levelsof cY in the plasma DNA are seen at this time (FIG. 3). The results inFIG. 3 suggest that the overall levels of cell-free DNA in the plasmaare not diagnostic of organ failure and do not track the“donor-specific” DNA signal

Similar trends were observed for another patient that had cY levelsincreasing at 5 months after transplant when a biopsy detected a grade3A rejection (FIG. 4). The percentage of cY (or % “Donor”) DNA isincreasing before and highest at rejection time. Like above, the amountof total cell-free DNA does not seem diagnostic for heart rejection

Collectively, these results establish that for heart transplantpatients, donor-derived DNA present in plasma can serve as a potentialmarker for the onset of organ failure.

Example 2: Genotyping of Transplant Donor and Transplant Recipient

FIG. 5 shows a general strategy to monitor all transplant patients.Genotyping of donor and recipient can establish a single nucleotidepolymorphism (SNP) profile for detecting donor DNA. Shotgun sequencingof cell-free DNA in plasma, with analysis of observed unique SNPs,allows quantitation of % Donor DNA in the sample. While any single SNPmay be difficult to detect with so little DNA in plasma, with hundred ofthousands or more signals to consider, high sensitivity should bepossible

Libraries of mixed genotypes can be created using two CEU (Mormon, Utah)HapMap lines. Approximately 1.2 million total variations between thesetwo individuals were already established using existing genotypingplatforms (e.g., Illumina Golden Gate). Usable SNPs must be homozygousfor the recipient and ideally homozygous for the donor as well. UsableSNPs comprise: (i) approximately 500,000 heterozygous donor SNPs (countwill be ½ of total donor fraction), (ii) approximately 160,000homozygous donor SNPs.

Sequencing Results: 4 lanes of Illumina sequencing are used to compare 4different levels of substitution of Donor DNA into Recipient DNA (SeeFIG. 6). Error rate of sequencing is currently ˜0.3-0.5% for basesubstitution. The use of quality scores for improved filtering of SNPcalls, or the use of resequencing, should reduce error rate and increasesensitivity. The use of more SNP locations (from full genotyping) shouldalso improve yield of signal with no change in protocol.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of preparing a cell-free nucleic acidfraction for analyzing transplant-derived cell-free nucleic acids in abiological sample from a solid organ transplant recipient, the methodcomprising: (a) providing a biological sample from the solid organtransplant recipient after the solid organ transplant recipient hasreceived a solid organ transplant from a solid organ transplant donor,(b) isolating a fraction from the biological sample from the solid organtransplant recipient to obtain an isolated fraction, wherein theisolated fraction comprises cell-free nucleic acids from the solid organtransplant, (c) extracting nucleic acids from the isolated fraction toobtain extracted nucleic acids, wherein the extracted nucleic acidscomprise cell-free nucleic acids from the solid organ transplant, (d)generating prepared extracted solid organ transplant-derived cell-freenucleic acids by preparing the extracted nucleic acids for ahigh-throughput sequencing reaction (HTS) or a digital polymerase chainreaction (dPCR), (e) analyzing transplant-derived cell-free nucleicacids by detecting and quantifying a homozygous or a heterozygous SNPwithin the transplant-derived cell-free nucleic acids usinghigh-throughput sequencing reaction (HTS) or digital polymerase chainreaction (dPCR).
 2. The method of claim 1, wherein the biological sampleis selected from the group consisting of blood or urine.
 3. The methodof claim 1, wherein the solid organ transplant received by the solidorgan transplant recipient is selected from the group consisting of: akidney transplant, a heart transplant, a liver transplant, a lungtransplant, a skin transplant, an intestine transplant, a pancreastransplant, a pancreas after kidney transplant, a simultaneouspancreas-kidney transplant.
 4. The method of claim 1, wherein thehomozygous or the heterozygous SNP is a nucleotide insertion, a repeat,or a deletion.
 5. The method of claim 1, wherein the homozygous or theheterozygous SNP has at least two alleles and allele frequency of 0.1 orgreater.
 6. The method of claim 1, wherein the homozygous or theheterozygous SNP is selected from a SNP profile that was generated bygenotyping the solid organ transplant recipient.
 7. The method of claim1, wherein the homozygous or the heterozygous SNP is selected from a SNPprofile that was generated by genotyping the solid organ transplantdonor.
 8. The method of claim 1, further comprising a step ofadministering an immunosuppressant treatment based on the analyzedtransplant-derived cell-free nucleic acids in the biological sample. 9.The method of claim 1, wherein an amplification reaction is performed onthe cell-free nucleic acids in the isolated fraction prior to analyzingtransplant-derived cell-free nucleic acids.
 10. The method of claim 1,wherein the cell-free nucleic acids comprise DNA, RNA, mRNA, miRNA,double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins,DNA/RNA hybrids, RNA hairpins, organ-specific RNA transcripts, or acombination thereof.
 11. The method of claim 1, wherein the cell-freenucleic acids comprise DNA.
 12. The method of claim 1, wherein thehigh-throughput sequencing reaction is a sequencing-by-synthesisreaction.
 13. The method of claim 1, wherein the biological sample isblood.
 14. The method of claim 1, wherein the biological sample isurine.
 15. The method of claim 1, wherein the solid organ transplant isa heart transplant.
 16. The method of claim 1, wherein the solid organtransplant is a lung transplant.
 17. The method of claim 1, wherein thesolid organ transplant is a kidney transplant.
 18. The method of claim1, wherein the solid organ transplant is a liver transplant.
 19. Themethod of claim 1, wherein the solid organ transplant is a pancreastransplant.